JPS6188873A - Preparation of transformed microorganism and l-tryptophan - Google Patents

Abstract

PURPOSE:To obtain a microbial strain having high productivity of L-tryptophan, by inserting the L-tryptophan biosynthesis gene of chromosome DNA of microorganism having L-tryptophan productivity into the chromosome DNA of a host cell belonging to Bacillus genus. CONSTITUTION:The fragment of a chromosome DNA of a microorganism having L-tryptophan productivity, and containing the genetic information essentially controlling the biosynthesis of L-tryptophan (e.g. the DNA having a molecular weight of about 5 M-Dalton and inserted into one of the incision points of plasmid PSDY3051 in plasmid PSDY3261 with ECORI) is inserted directly to a chromosome DNA of a host microorganism belonging to Bacillus genus.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a microorganism whose ability to produce L-tryptophan has been enhanced through transformation, and a method for producing sti-tryptophan using the microorganism.

[Prior art and problems to be solved by the invention] [-
Tryptophan is an important compound as a type of essential amino acid, and as it is a substance that is particularly susceptible to nutritional deficiencies, there is a strong desire to develop an economical method for producing it, and for this reason, various methods have been proposed. However, the fermentation method has attracted particular attention, especially when the L.
-Improvement of tryptophan-producing microorganisms is an important issue.

Conventionally, artificial mutation methods have generally been used to improve substance-producing microorganisms, but none have been found to be fully satisfactory from the viewpoint of L-tryptophan productivity. In addition, attempts have been made to improve microorganisms using recently developed genetic recombination techniques, but they have not yet reached the level of success for industrial use.

In general, the improvement of microorganisms through genetic modification techniques involves first removing the gene from its donor cell, and then converting it into vector DNA in vitro.
A and the resulting recombinant DNA is incorporated into host cells. Then, host cells containing the desired recombinant DNA are grown, and then the introduced gene is expressed to obtain 1 q of the desired product. As vectors used in this case, plasmids or bacteriophages are generally known. However, although multiple plasmids exist within a cell and are highly effective in gene amplification, they are generally unstable within the host bacterium and are prone to shortening and falling off, so unless a method for stabilizing them within the host bacterium is established, Not suitable for large-scale fermentation production. On the other hand, when bacteriophage is used as a vector, the introduced gene is integrated into the chromosomal DNA and is therefore quite stable within the host bacterium.On the other hand, this method has limitations such as a limited host range, making it difficult to improve the bacterium. There are problems in using it.

Therefore, the present inventors have proposed that a wide range of host bacteria can be used, and the gene to be introduced can be directly transferred to the host without using vector DNA such as a plasmid or bacteriophage so that the introduced gene can stably exist within the host bacteria. We have established a method for transformation by insertion (integration) into the chromosomal DNA of bacteria, and have conducted various studies to apply this method to obtain microorganisms with high L-tryptophan production, resulting in the present invention.

c. Means for Solving Problems] According to the present invention, a portion (hereinafter referred to as T r
It is called the pm gene. ) into the chromosomal DNA of a host bacterium selected from microorganisms belonging to the genus Bacillus without using vector DNA such as a plasmid or bacteriophage. The Ll-Liftfan high-producing microorganism having Ll-Liftfan and the Trp gene further linked with a selectable marker gene are inserted into the chromosomal DNA of a host bacterium to obtain the desired L-tributofan. The present invention provides microorganisms that allow easy selection of highly productive microorganisms.

According to the present invention, the L-t-libhiphan-producing microorganism obtained by these transformations is further cultivated in J8Ja to produce L-tryptophan in the culture, and the L-tryptophan is collected. Provided is a method of producing L-tryptophan in an economically advantageous manner.

Below, the microorganism of the present invention, the 14 methods for its preparation, and the method for producing L-tryptophan using the same will be explained in more detail.

The T rpi gene used in the present invention is usually excised using an appropriate restriction enzyme from the chromosomal DNA of a microorganism capable of producing L-tryptophan, but it has high homology with the chromosomal DNA of the host bacterium. In principle, there are no particular restrictions on the origin of the plant, including wild strains isolated from soil and other natural products that have the ability to produce L-tryptophan, as well as those that have been exposed to ultraviolet rays or chemically. Artificial mutant strains obtained by treatment with biochemicals or recombinant D containing genetic information for regulating the biosynthesis of L-tryptophan II'7 produced using genetic recombination technology.
Any NA etc. is fine. In this case, it is preferable that the Trp gene consists of only a part that has genetic information that regulates the biosynthesis of l-tryptophan and does not contain any other unnecessary parts, but depending on the type of restriction enzyme used, In addition to the Tr11 gene, some other parts may be included before and after the Tr11 gene, and even such parts may have a negative impact on the homology with the host bacteria and the biosynthesis of the target L-t-ributofun7. It can be used unless Further, the Trp gene does not need to have all of the genetic information that regulates the biosynthesis of L-tryptophan, and a DNA containing only a portion of the genetic information can also be used.

Examples of microorganisms having chromosomal DNA containing the Tr11 gene include Bacillus amyloliquefacens, Bacillus amylolyticus, Bacillus alcalophilus, Bacillus coagulans, Bacillus licheniformis, Bacillus narratou, and Bacillus narratou.・
Microorganisms belonging to the genus Bacillus, such as Bacillus subtilis and Bacillus stearothermophilus, and their mutant strains;
Strains bred by genetic recombination using these parent strains are listed. In addition, recombinant DNA obtained by processing these chromosomal DNAs with the chromosomal DNAs of other microorganisms, plasmids, bacteriophages, and other DNAs using genetic engineering techniques.
NA is raised. In addition, there are no particular restrictions on the restriction enzymes used to excise the Trp gene from these DNAs, but it is desirable to have fewer cleavage sites in the Trp gene, such as EcoRI, BamHI, Sal
I, 3ac engineering, pvu [, XhO ■, Xba engineering, Xb
oI.

Examples include MIIJ ■.

In principle, any host strain into which the Trp gene should be introduced may be used as long as its chromosomal DNA has homology to the T rpm gene, but in practice, it is possible to introduce the Trp gene using the fermentation method, which is the original purpose of the present invention. It is necessary that the microorganism is suitable for producing L-tryptophan, and from this point of view, pathogenic microorganisms and those that are difficult to manage or cannot be used for industrial use are excluded. Practically speaking, microorganisms belonging to the genus Bacillus are used, which are easy to culture and have a track record of actual industrial use.
If the same type of microorganism as the one from which the r113iff gene was extracted is used as a host bacterium, so-called self-cloning will occur, and even if the transformed bacterium undergoes genetic manipulation, the recombinant DNA will not be produced.
There are no restrictions in terms of experimental guidelines, which is convenient. The host bacteria used in the present invention are preferably bacteria in which the microorganism's inherent tributophane biosynthesis feedback suppression mechanism has been released by producing and accumulating tryptophan, such as tryptophan analogs (5-fluorotryptophan, 5-methyltryptophan). etc.) are resistant bacteria. Typical examples include, for example,
8, 49-20391, 49-85289, 51
-64921, 53-1358, 53-39517
, 56-92796, 58-94391, 58-
107190, 58-107193, 58-107
194, 58-107195, 58-138389
, 58-220693, 59-120091, 5
Examples include bacteria described in 9-130181 and the like.

Transformation of the Trp3] gene into the chromosomal DNA of the above-mentioned host bacterium can be carried out using known methods, for example, the combination cell method (J, Bacterial 81.741 (1999)).
61)] or the protoplast method (Molec, ge
n.

Qenet, 168. ．． 111 (1979))
etc. In addition, for transformation, Trp
In order to perform the insertion (integration) of the m gene into the host bacterial chromosomal DNA, it is necessary to make the T rpm gene into a circular form using an enzyme such as T4 ligase, and to further increase the frequency, the Trp gene must be sufficiently concentrated in advance. It is preferable to keep it. In addition, in order to confirm the presence or absence of transformation, we used strains that have become tryptophan auxotrophic due to a partial mutation of the Drp gene as host bacteria (recipient bacteria), such as Bacillus 5O-
53 (JP 59-170047), AJ11712
Transformed strains can be selected as non-tryptophan auxotrophs by using JP-A-58-89194), UOT 0531 (Institute of Applied Microbiology, University of Tokyo), and the like. However, if a strain in which the gene encoding the rate-limiting enzyme in the tryptophan biosynthesis pathway is mutated, the efficiency of improving the tryptophan productivity of the transformed strain will decrease, so the gene encoding the rate-limiting enzyme will be mutated. It is desirable to use an unmutated tryptophan auxotrophic strain.

However, these transformations are treatments for confirming the presence or absence of transformation, and confirmation methods are not necessarily limited to these methods. In order to select the target bacteria from the cells produced by transformation, the transformed bacteria must be
As a result of the pa gene being inserted into the trochanter, tryptophan non-requirement (
Taking advantage of the fact that the L-tryptophan productivity was increased as a result of the amplification of the T r9') M trochanter, the culture medium to which anthranilic acid was added was The L-tryptophan productivity in the strain is investigated to obtain a strain with improved L-tryptophan productivity.

Furthermore, due to recombination between the tryptophan gene introduced at the time of transformation and the missing part of the chromosomal DNA, transformation that becomes tryptophan non-auxotrophic occurs, but in this case, the trochanter of the T rpiu gene does not occur. In the above case, the frequency of such transformation is relatively high, and T rp
Selection of amplified transformed strains of 312 Butsuko takes time and effort.

In order to improve this drawback, it is possible to use a Trtlfl gene linked with a selectable marker gene.

The selectable genetic marker used in the method of the present invention may be any gene marker as long as it is expressed within the host bacterium.
- Examples include: Examples of drug resistance genes include chloramphenicol resistance, tetracycline resistance, and erythromycin resistance. ,
These include kanamycin resistance, neomycin resistance, streptomycin resistance, penicillin resistance, and phosphomycin resistance.

The method for obtaining highly productive L-tryptophan microorganisms of the present invention will be described below in more detail by showing typical examples. However, these are merely examples, and the present invention is not limited to these.

The chloramphenicol resistance gene of plasmid pTP4 was cloned into phage ρ11 DNA by a conventional method, and then the phage DNA was treated with restriction enzyme EC0R.
Plasmid 1) BR322 DNA cut with I and pre-cut with EC0RI is mixed at a temperature such that the number of ends of the resulting DNA fragments is the same, and a ligation reaction is performed using T4 phage ligase. make me wake up This DN
Escherichia coli C600tr treated with calcium chloride using A
p.

The leu, thr, rlr, and mk- strains were transformed using a conventional method to obtain Escherichia coli 5D-1007 (Feikoken Accession No. 7860), which is resistant to chloramphenicol, ampicillin, and tetrazycline. Obtained.

Plasmid of the transformed bacterium (referred to as psD3165)
An approximately 2.5 megadalton chloramphenicol resistance gene was inserted into the EcoPI breakpoint of DBR322.

Next, Bacillus subtilis plasmid pTA1015 was treated with restriction enzyme Eco.
E. coli 5D cut with RI and the same <EcoRI cut
-1007 plasmid DSD3165 and the resulting DNA fragments are mixed at a concentration such that the numbers of ends are the same, and a ligation reaction is caused using T4 phage ligase. Using this DNA, Bacillus 5D30
-130181) was transformed by the competent cell method to obtain a strain resistant to chloramphenicol.

Plasmids were separated from these transformed strains and a restriction enzyme map was prepared, and a plasmid having a restriction enzyme map as shown in FIG. 1 was obtained. This plasmid psDY
The transformed strain Bacillus 5D-1006 containing Bacillus 3051 has been deposited as Bacillus 7859. Plasmid psDY3051 of the transformed bacterium had a chloramphenicol resistance gene of about 2.5 megadaltons inserted at the EC0RI breakpoint of pTΔ1015.

Next, psDY3051 was partially cleaved with the restriction enzyme EC0RI, and the cloned phage φ105DNASDP-12 containing the tryptophan operon derived from i-ributophane analog resistant Bacillus subtilis (Japanese Patent Application Laid-open No. 1993-111125)
892) is also cleaved with a restriction enzyme (EcoRI), the two DNAs are mixed, and ligated using T4 phage ligase. Using this DNA, Bacillus subtilis BD22
4 (trpC2thr-5rec E4) by the competent cell method to obtain transformed bacteria that are resistant to chloramphenicol and exhibit no requirement for Trp.

The recombinant plasmid was isolated and purified from the transformed bacteria by a conventional method, and a restriction enzyme map was prepared.A plasmid having a restriction enzyme map as shown in FIG. 2 was obtained. This plasmid (referred to as pSDY3261) contains psDY3051
About 5 megadaltons of DNA at one of the EC0RI breakpoints of
had been inserted. This insert DNA is used for each weighing tryptophan auxotroph (trD A, tri) B, trl)
C, trII D or trp E mutant strains)
When psDY3261 was used as a recipient strain and psDY3261 was used as a donor DNA, Trp-non-auxotrophic transformed bacteria frequently appeared in all of them, which is thought to determine the genetic information that regulates the biosynthesis of tryptophan.

Plasmid DNAΔpsDY3261 was digested with restriction enzyme ECOR.
The fragments are separated by agarose gel electrophoresis. The part of the agarose gel containing the DNA fragment containing the genetic information that regulates tryptophan biosynthesis was cut out, and the NA was separated using a conventional method. Ko (7)
T4 DNA ligase is applied to the DNAflJi piece to recombine it and form a circular DNA. Using this DNA, anthranilate synthetase-deficient strain Bacillus 5D-53 of Bacillus amyloliquifacens IAM1521 strain Yamaki was prepared.
(Japanese Patent Application No. 59-170047) and select an anthranilic acid non-requiring strain. These anthranilic acid non-auxotrophic transformed strains were cultured in the ground in liquid 1a containing anthranilic acid, and strains with improved L-1-ributophane productivity were selected and transformed into Bacillus 5D-1004 (FEI accession number No. No. 7857) was obtained.

The mycological properties of this bacterium are essentially the same, except that the original strain has Batisur 5D-53 and an anthranilic acid auxotrophy, and T rpa Butsuko has been amplified, resulting in a high L-t-ributophane production ability. be.

L-t of the obtained transformed bacterium Bacillus 5D-1004
- The results of measuring ributofin synthase activity are as follows.

Bacterium L-tryptophan synthetase specific activity Bacillus 5D-53100 Bacillus 5D-1004130 For these bacteria, Subizizene minimum medium ((Nl-14) in the presence of anthranilic acid (80 ppm))
2 304 0.2% K 2 tlPO41
4% KH2PO40.6% Sodium citrate ・
2112 0 0.1% M(l
SO471-1200.02% Glucose 0.5%
The results of 1-)-ributofun accumulation when cultured at 37°C for 1.5 hours are shown.

Bacterial strain L-tryptophan storage W4 (μ/4) Bacillus 5D-5332 Bacillus 5D-100443 In addition, in order to facilitate the selection of bacterial strains with improved L-tryptophan productivity by the above method, the following There is an improved method.

In the same manner as above, a DNA fragment having genetic information for regulating tryptophan biosynthesis and a DNA fragment having genetic information for chloramphenicol resistance are separated and purified,
Both are mixed and T4 DNA ligase is applied to create a circular DNA having genetic information for regulating tryptophan biosynthesis and genetic information for chloramphenicol resistance. Create this DNA. Using this DNA, I
The 5-fluorotryptophan-resistant strain Bacillus amyloliquifaciens 5D-30 (see Japanese Patent Application Laid-open No. 130181/1981) derived from the AM1521 strain is transformed, and chloramphenicol-resistant transformants are selected. These chloramphenicol-resistant transformants were cultured in a liquid medium containing anthranilic acid, and strains with improved L-t-ributophane productivity were selected and Bacillus 5D-1003
(Feikoken accession number No. 7856) was obtained.

The mycological properties of this rAa are substantially the same as those of the original strain Bacillus amyloliquefacens 5D-30, except that it is chloramphenicol resistant and has a high L-tryptophan production ability.

The measurement results of L-tryptophan synthetase activity of the obtained transformed bacterium Bacillus 5O-1003 are as follows.

Bacillus L-tryptophan synthetase active Bacillus 5D-30100 Bacillus 5D-1003175 These bacteria were cultured on a minimum amount of spizizen in the presence of anthranil (80 ppm) <37°C, 1.
The results of L-tryptophan accumulation after 5 hours) are shown.

Bacterial strain L-1-ributophane accumulation (μ'j
/mQ) Bacillus amyloliquefaciens 5D-3034 Bacillus 5D-100358 According to the method of the present invention, L-tryptophan can be produced by culturing Bacillus 5D-1003 or Bacillus 5D-1004 on borrowed land containing anthranilic acid or its salt. can be generated. There is no particular limit to the concentration of anthranilic acid or its salt in the nutrient medium, but it is generally 0.1 from the viewpoint of the desired yield of L-tryptophan, culture conditions, and economics.
The concentration should be ~3000 η/a, preferably 100 to 1000 η/a.

The leased land that can be used in the method of the present invention may be any land as long as the microorganisms can be grown by culturing.For example, carbon sources include grapes, molasses, sucrose, n powder, and starch saccharification. As nitrogen sources, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphorus, urea, nitric M salts, etc. are used as appropriate. . Examples of inorganic salts include salts of phosphoric acid, potassium, magnesium, manganese, etc., such as ammonium phosphate, potassium rAate,
gJl sodium, magnesium sulfate, ferrous Ta acid,
Industrial chemicals such as manganese sulfate and caustic potash may be used, and other trace elements such as calcium, zinc, copper, cobalt,
Salts such as molybdenum may be added, and vitamins, amino acids, nuclear M-related substances, etc. are not particularly necessary for the growth of bacteria, but they may be added, meat extract, yeast extract, etc. ] ~ Organic substances such as steep liquor and peptone may be added. Anthranilic acid lotus 1~
It may be added as an aqueous solution of lithium 1Bs potassium salt, ammonium salt, etc., or an ethanol or methanol solution of the free acid.

Cultivation in the method of the present invention can be carried out under an aerobic tray 1, for example, by aerated stirring or reciprocating shaking. Culture conditions are not particularly limited, but generally speaking, temperature is 30-45°C, pHG, O-8.0, and 15-60R.
It will be carried out under conditions of about

The desired L-t-ributophane can be collected from the culture solution or culture according to a conventional method. For example, the culture solution is centrifuged, and the supernatant is treated with an ion exchange resin, activated carbon, etc. in an appropriate combination for 1-t.
- A ribtofan can be operated in single action.

EXAMPLES The present invention will be described in detail below, but it goes without saying that the scope of the present invention is not limited to these Examples.

Example 1 Glucose 5%, ammonium sulfate 0.2%, K21-1PO4+1
．． 4%, KH2PO40.6%, sodium citrate.
2+-1201'j, M(] SO0・71-1200
．． 02%, f-esO4・71-120 1F) pHl
Liquid material (1) l-17 containing and MnSO4lppm
, 0) Add 800 ppm of anthranil H to 2 fL,
The Bacillus 5D1003 obtained above was inoculated into this, and the temperature was kept at 35°C.
Culture with aeration and agitation in a 51 jar fermenter.

During the culture, when the anthranilic acid concentration decreased to 50 ppm, additional addition was made as appropriate so that the anthranilic acid concentration was approximately 11,000 ppm, and glucose was added at 10 ppm during the culturing.
By adding 0g and further adding ammonia water, the p was doubled.
The cells were cultured for 15 hours while maintaining H at 7.0±0.4. The accumulation of tryptophan was 9.2 g/l in parallel operation of Bacillus Ami O Liquidaciens 5D-30.
It showed 1.97 times that of the stock.

Actual example 2 Glucose 5%, ammonium sulfate 02%, K21-(PO414%
, KH2PO40.6%, sodium citrate 2H2
01g, MgSO4・7H200,02%, FeS
O4・7H20lppm, MnS 04 1p
l) 800 ppm of Ni7-ntranyl W (pH 7,0>2a) was added, and the transformed bacterium Bacillus 5D1004 was inoculated thereto, and the mixture was cultured with aeration and stirring at 35° C. in a 5-year jar fermenter.

Anthranil in cultivation! ≠ When the anthranilic acid concentration decreases to about 100 opa
Further, 1009 glucose was added during the cultivation, and the culture was continued for 15 hours while maintaining the doubling pH at 7.0±0.4 by adding ammonia water. The accumulation of L-1-ributophane was approximately 1.66 times higher than that of the host bacteria run in parallel at 7.3 g/l.

[Brief explanation of the drawing]

FIG. 1 shows the restriction enzyme map of plasmid p'S DY 3051, and FIG. 2 shows the restriction enzyme map of plasmid f) SDY3261, and the meanings of symbols follow the conventions used in the field of genetics.

Claims (5)

[Claims] (1) A portion of the chromosomal DNA of a microorganism capable of producing L-tryptophan containing genetic information that substantially regulates the biosynthesis of L-tryptophan is inserted into the chromosomal DNA of a host bacterium selected from microorganisms belonging to the genus Bacillus. Tenaru L
-Tryptophan-producing microorganisms. (2) A drug resistance gene, an amino acid biosynthesis gene other than L-tryptophan, or another An L-tryptophan-producing microorganism obtained by linking a selectable marker gene and inserting this into the chromosomal DNA of a host bacterium selected from microorganisms belonging to the genus Bacillus. (3) The L-tryptophan-producing microorganism according to claim 1 or 2, wherein the host bacterium has tryptophan analog resistance. (4) A portion of the chromosomal DNA of a microorganism capable of producing L-tryptophan that has genetic information that substantially regulates the biosynthesis of L-tryptophan, or a drug resistance gene, a biosynthetic gene for amino acids other than L-tryptophan, or An L-tryptophan-producing microorganism obtained by inserting a gene linked with another selectable marker gene into the chromosomal DNA of a host bacterium selected from microorganisms belonging to the genus Bacillus is cultured in a culture medium, and L- A method for producing L-tryptophan, which comprises producing tryptophan and collecting it. (5) L- according to claim 4, wherein the medium is a medium containing anthranilic acid or a salt thereof.
Method for producing tryptophan.